Evaluation of the intervertebral neck injury criterion using simulated rear impacts

The Intervertebral Neck Injury Criterion (IV-NIC) is based on the hypothesis that intervertebral motion beyond the physiological limit may injure spinal soft tissues during whiplash, while the Neck Injury Criterion (NIC) hypothesizes that sudden changes in spinal fluid pressure may cause neural injury. Goals of the present study, using a biofidelic whole cervical spine model with muscle force replication, were to correlate IV-NIC with soft-tissue injury, determine the IV-NIC injury threshold, and compare IV-NIC and NIC. Using a bench-top apparatus, rear-impacts were simulated at 3.5, 5, 6.5, and 8 g horizontal accelerations of the T1 vertebra. Pre- and post-whiplash flexibility tests measured the soft tissue injury threshold, i.e. significant increases in the intervertebral neutral zone (NZ) or range of motion (ROM) above corresponding baseline values. Extension IV-NIC peaks correlated well with NZ and ROM increases at C0-C1 and at C3-C4 through C7-T1 (r=0.64 and 0.62 respectively, p<0.001). Average IV-NIC injury thresholds (95% confidence limits) varied among the intervertebral levels and ranged between 1.5 (1.1, 1.9) at C5-C6 and 3.4 (2.4, 4.4) at C7-T1. The NIC injury threshold was 8.7 (7.7, 9.7) m2/s2, substantially less than the proposed threshold of 15 m2/s2. Results support the use of IV-NIC for determining the cervical spine injury threshold and injury severity. Advantages of IV-NIC include the ability to predict the intervertebral level, mode, severity, and time of the cervical spine soft-tissue injury.     

Dynamic changes in expression of heme oxygenases in mouse heart and liver during hypoxia

Heme oxygenase cleaves heme to form biliverdin, carbon monoxide (CO), and iron, and consists of two structurally related isozymes, HO-1 and HO-2. HO-2 is also known as a potential oxygen sensor. Here we show that the relative CO content in arterial blood, which reflects the total amount of endogenous heme degradation, dynamically changes in mice during acclimatization to normobaric hypoxia (10% O2), with the two peaks at 1 day and 21 days of hypoxia. The expression levels of HO-1 and HO-2 proteins were decreased by 20% and 40%, respectively, in the mouse liver at 7 days of hypoxia, which returned to the basal levels at 14 days. On the other hand, HO-1 and HO-2 proteins were increased 2-fold and 1.3-fold, respectively, in the heart at 28 days of hypoxia. Thus, hypoxia induces or represses the expression of HO-1 and HO-2 in vivo, depending on cellular microenvironments.     

Role of afferent pathways of heat and cold in body temperature regulation

The detection of surface and internal temperatures is achieved by axons terminating at lamina I of the spinal dorsal horn, otherwise approached only by nociceptive afferents. Recent advances in thermal physiology research have disclosed that temperature-sensitive ion channels belonging to the transient receptor potential family exist in the peripheral sensory neurons and in the brain. Thermosensory, nociceptive and polymodal afferents project to different thalamic nuclei, and specific pathways to the insular cortex evoke the conscious experience of thermal sensation. The posterior insular region represents discriminative thermal sensation, while the largest correlation with subjective ratings of temperature is located in the orbitofrontal and anterior insular cortex. The insular cortex forms an integrative part of the limbic system and is closely tied with the hypothalamus, the amygdala, the anterior cingulate cortex and the orbitofrontal cortex and emerges as the main coordinator of behavioral, autonomic and endocrine responses to both non-noxious and noxious thermal stimuli. The firing rate of warm and cold receptors is not altered by pyrogens. A strong correlation between the onset of fever and production of superoxide by macrophages following the injection of pyrogens implicates reactive oxygen species as elicitors of fever, a hypothesis strengthened by the observation that oxygen radical scavengers or thiol reductants act as antipyretics. Oxidative stress appears to be sensed by the brain and a likely structure for its detection may be the redox-sensitive site of the N-methyl-d-aspartate (NMDA) receptor for glutamate, in that oxidation of this site causes fever while its reduction lowers body temperature, effects which are abrogated by specific NMDA receptor blockers.     

Mapping of functional sites on the primary structure of the contractile tail sheath protein of bacteriophage T4 by mutation analysis

In order to determine the functional roles of amino acid residues in gp18 (gp: gene product), the contractile tail sheath protein of bacteriophage T4, the mutation sites and amino acid replacements of available and newly created missense mutants with distinct phenotypes were determined. Amber mutants were also utilized for amino acid insertion by host amber suppressor cell strains. It was found that mutants that gave rise to a particular phenotype were mapped in a particular region along the polypeptide chain. Namely, all amino acid replacements in the cold-sensitive mutants (cs, which grows at 37 degrees C, but not at 25 degrees C) and the heat-sensitive mutant (hs, lose viability by incubation at 55 degrees C for 30 min) except for one hs mutant were mapped in a limited region in the C-terminal domain. On the other hand, all the temperature-sensitive mutants (ts, grow at 30 degrees C, but not at 42 degrees C) and carbowax mutants (CBW, can adsorb to the host bacterium in the presence of high concentrations of polyethylene glycol, where wild-type phage cannot) were mapped in the N-terminal protease-resistant domain, except for one ts mutant. The results suggested that the C-terminal region of gp18 is important for contraction and assembly, whereas the N-terminal protease-resistant domain constitutes the protruding part of the tail sheath.     

Enzymatic properties of pierisin-1 and its N-terminal domain, a guanine-specific ADP-ribosyltransferase from the cabbage butterfly

The cabbage butterfly, Pieris rapae, produces an ADP-ribosylating cytotoxic protein, pierisin-1. Unlike other ADP-ribosylating toxins, the acceptor site for ADP-ribosylation by pierisin-1 is the N-2 position of guanine bases in DNA. The present study was designed to characterize this novel guanine-specific ADP-ribosyltransferase, pierisin-1. The N-terminal polypeptide from Met-1 to Arg-233, but not the C-terminal Ser-234-Met-850 polypeptide, was found to exhibit guanine ADP-ribosyltransferase activity. Trypsin-treated pierisin-1, which is considered to be a "nicked" full-length form composed of associated N- and C-terminal fragments, also demonstrated such activity. Optimum conditions for the N-terminal polypeptide of pierisin-1 were pH 8-10, 37-40 degrees C, in the presence of 100-200 mM NaCl or KCl. Other metal ions such as Ca(2+) or Mg(2+) were not required. Kinetic studies demonstrated potent ADP-ribosyltransferase activity with a K(M) value for NAD of 0.17 mM and k(cat) of 55 per second. Under these optimum conditions, the specific activity of trypsin-treated pierisin-1 was about half (k(cat) = 25 per second). When the conditions were changed to pH 5-7 or 10-20 degrees C, some activity (6-55% or 5-20%, respectively, of that under optimal conditions) of the N-terminal polypeptide was still evident; however, almost all of the trypsin-treated enzyme activity disappeared. This implies the inhibition of the N-terminal enzyme domain by the associated C-terminal fragment. Long-term reactions indicated that a single molecule of pierisin-1 has the capacity to generate more than 10(6) ADP-ribosylated DNA adducts, which could cause the death of a mammalian cell.     

The N-terminal region of NTAK/neuregulin-2 isoforms has an inhibitory activity on angiogenesis

NTAK (neural- and thymus-derived activator for ErbB kinases), also known as neuregulin-2, is a member of the epidermal growth factor (EGF) family, which binds directly to ErbB3 and ErbB4 and transactivates ErbB2. Because ErbB signaling has been implicated in various angiogenic mechanisms, the effect of NTAK (which has at least nine isoforms due to alternative splicing) in angiogenesis is explored. One isoform, NTAKgamma, inhibited cell growth in terms of DNA synthesis and cell numbers in vascular endothelial cells specifically, whereas NTAKalpha and beta had no activity. On the other hand, NTAKgamma secreted by transfected MDA-MB-231 cells inhibited endothelial cell growth, and NTAKgamma expressed in endothelial cells by adenovirus infection suppressed cell growth in a dose-dependent manner. The EGF-like domain of NTAKgamma did not have this activity. The NTAKdelta isoform, which had the Ig-like domain but not the EGF-like domain, inhibited proliferation of endothelial cells. NTAKdelta prevented hyper-phosphorylation of the retinoblastoma tumor suppressor protein and caused G(1) arrest in endothelial cells. Both NTAKgamma and delta isoforms displayed anti-angiogenic activity in the chick embryo chorioallantoic membrane in vivo. These results suggest that the active site of NTAK is localized outside of the EGF-like domain but within the N-terminal region, including the Ig-like domain, of NTAK.     

Neuroglycan C, a novel member of the neuregulin family

Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan expressed predominantly in the brain that possesses an EGF-like extracellular domain. The goal of the present study was to determine whether NGC may activate ErbB tyrosine kinases. A recombinant human NGC extracellular domain induced tyrosine phosphorylation of ErbB2 and ErbB3 as well as cell growth of the human breast tumor cell lines, T47D and MDA-MB-453. In vitro pull-down assay revealed that NGC could directly bind to a recombinant ErbB3-immunoglobulin Fc fusion protein (ErbB3-Fc) but not to ErbB1-Fc, ErbB2-Fc or ErbB4-Fc. A newly established anti-ErbB3 neutralizing monoclonal antibody (#5C3) almost completely blocked NGC-induced ErbB activation in MDA-MB-453 cells. Taken together, these data indicate that NGC is an active growth factor and a direct ligand for ErbB3 and that NGC transactivates ErbB2. Thus, NGC should be classified as the sixth member (neuregulin-6) of the neuregulin family.